It is now possible through the techniques of genetic engineering to cause a host cell to produce "heterologous" polypeptides, i.e., polypeptides which are not naturally produced by that species of cell. A variety of mammalian polypeptides have been produced in E. coli such as somatostatin, described by Itakura et al., Science 198:1056 (1977); the component A and B chains of human insulin, disclosed by Goeddel et al., Proc. Natl. Acad. Sci. USA 76:106 (1979); human growth hormone, disclosed by Goeddel et al., Nature 281:411 (1980); human fibroblast interferon, disclosed by Goeddel et al., Nucleic Acids Res. 8:4057 (1980); and human serum albumin, as disclosed by Lawn, et al., Nucleic Acids Res. 9:6103 (1981).
In the application of current recombinant DNA procedures, specific DNA sequences are inserted into an appropriate DNA vehicle, or vector, to form recombinant DNA molecules that can replicate in host cells. Circular double-stranded DNA molecules called plasmids are frequently used as vectors, and the preparation of such recombinant DNA forms entails the use of restriction endonuclease enzymes that can cleave DNA at specific base sequence sites. Once cuts have been made by a restriction enzyme in a plasmid and in a segment of foreign DNA that is to be inserted, the two DNA molecules may be covalently linked by an enzyme known as a ligase. General methods for the preparation of such recombinant DNA molecules have been described by Cohen et al. [U.S. Pat. No. 4,237,224], Collins et al. [U.S. Pat. No. 4,304,863] and Maniatis et al. [Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Habor Laboratory].
Once prepared, recombinant DNA molecules can be used to produce the product specified by the inserted gene sequence only if a number of conditions are met. Foremost is the requirement that the recombinant molecule be compatible with, and thus capable of autonomous replication in, the host cell. Much recent work has utilized Escherichia coli as a host organism, because it is compatible with a wide range of recombinant plasmids. Depending upon the vector/host cell system used, the recombinant DNA molecule is introduced into the host by transformation, transduction or transfection.
Detection of the presence of recombinant plasmids in host cells may be conveniently achieved through the use of plasmid marker activities such as antibiotic resistance. For example, a host bearing a plasmid coding for the production of an ampicillin-degrading enzyme could be selected from unaltered cells by growing the host in a medium containing ampicillin. Further advantage may be taken of antibiotic resistance markers where a plasmid codes for a second antibiotic-degrading activity at a site where the selected restriction endonuclease makes its cut and the foreign gene sequence is inserted. Host cells containing the proper recombinant plasmids will then be characterized by resistance to the first antibiotic but sensitivity to the second.
The mere insertion of a recombinant plasmid into a host cell and the isolation of the modified host will not in itself assure that significant amounts of the desired gene product will be produced. For this to occur, the foreign gene sequence must be fused in proper relationship to a signal region in the plasmid called a promoter for DNA transcription.
Transcription of the gene into mRNA is instituted when an enzyme known as RNA polymerase contacts and interacts with the promoter sequence, thereafter moving along the gene and causing the synthesis of a mRNA molecule which is specified by the gene. The mRNA is thereafter translated into a specific polypeptide by cellular constituents, such as ribosomes, transfer RNAs, etc.
A variety of promoter sequences have been used to promote the expression of foreign or heterologous genes (i.e., genes not normally present) in E. coli, such as the lac promoter, Backman et al., Cell 13:65 (1978); the trp promoter, Hallewell et al., Gene 9:27 (1980); and the phage lambda P.sub.L promoter, Bernard et al., Gene 5:59 (1979).
A variety of gene promoter/operator (po) systems are "inducible"; i.e., their activity can be varied substantially by the presence or absence of a certain substance or condition. For example, the lac promoter system of E. coli has a relatively low level of activity in the absence of lactose. In this condition, it is said to be repressed. In the presence of an inducer such as lactose itself or isopropyl-.beta.-D-thiogalactoside (IPTG), however, the lac promoter system becomes derepressed and causes a high level of transcription of the gene sequence adjoining the promoter. See, e.g., J. Miller and W. Reznikoff, The Operon 2d Edition, Cold Spring Harbor Laboratory, New York (1982). Other inducible promoter systems include e.g., the trp promoter (which has relatively low activity if an excess of tryptophan is present and higher activity if a low concentration of tryptophan or 3-betaindolylacrylic acid is present; see Hallewell et al., supra), and the phage lambda P.sub.L promoter (which has a relatively low level of activity at about 30.degree. C. and higher activity at 41.degree. C. in the presence of a temperature-sensitive mutant lambda repressor; see Bernard, supra).
The promoter regions present in the DNA of bacteriophage T7 are of particular interest since bacterial cells infected by this bacteriophage almost immediately begin to produce only T7 bacteriophage polypeptides, to the virtual exclusion of the host cell's own requirements. This action is in large measure a consequence of a very strong interaction between the bacteriophage T7 promoters and the T7 RNA polymerase. Bacteriophage T7 DNA is also known to code for a protein kinase which inactivates E. coli RNA polymerase by phosphorylation, thereby reducing transcription of E. coli DNA. It is because of these two factors that the protein synthesizing machinery of E. coli is directed almost exclusively to the production of bacteriophage protein shortly after infection.
It would, therefore, be very useful if the methods utilized by bacteriophage T7 to exclusively direct synthesis of bacteriophage-required proteins could be harnessed to direct the synthesis of polypeptides which are desired for medical, diagnostic, research, and other purposes.
A suggestion of the use of bacteriophage T7 promoters to direct the transcription of a cloned gene in bacteria is contained in McAllister et al., J. Mol. Biol. 153:527 (1981). This reference postulates that the T7 RNA polymerase which would be required to initiate the T7 promoter directed transcription would have to be supplied to the cell by infection or from the cloned T7 polymerase gene. The reference is silent as to any specific method for achieving this result.
Supplying T7 RNA polymerase by infection would not be practical, since the enzyme is produced only briefly during infection and does not accumulate to high levels. Furthermore, T7 bacteriophage infection would be accompanied by natural competition of T7 RNA polymerase for the cloned gene promoter and promoter regions present in the bacteriophage DNA itself, and the infected cells would lyse within a short period of time.
The cloning and expression of the gene for T7 RNA polymerase (T7 Gene 1) is reported in a later paper [Davanloo et al., Proc. Natl. Acad. Sci. USA 81:2035 (1984)]. The authors caution, however, that the presence of any T7 promoters in a plasmid containing T7 Gene 1 in the same orientation as the T7 promoters would have to be scrupulously avoided. They theorized that T7 RNA polymerase, by transcribing completely around such a plasmid would be able to direct the synthesis of its own mRNA from the cloned fragment that contained both the promoter and the intact T7 Gene 1. Such a construction, they believe, would lead to an autocatalytic increase both in the level of T7 RNA polymerase and in the rate of transcription of the plasmid; a condition which they say would almost certainly be lethal to the cell.
Davanloo et al. state that a single molecule of active T7 RNA polymerase would potentially be sufficient to trigger this response so such a construction, i.e., a plasmid containing both a T7 promoter region and the T7 RNA polymerase gene, would be stable only if there was absolutely no expression of the cloned T7 RNA polymerase gene. This situation, they conclude, may be difficult or impossible to achieve. These authors, therefore, utilized a bacterial promoter sequence to direct the transcription of the T7 Gene 1; but were very careful to be sure that the plasmid construct did not contain an intact T7 promoter region. This is complicated to achieve since the bacteriophage DNA has no conveniently located restriction sites that will permit the ready isolation of a fragment which contains all of the T7 Gene 1 coding sequence without any promoter region.
Finally, Davanloo et al. state that it is unlikely that a sequence containing both the T7 Gene 1 translatable coding sequence and a T7 promoter region could be cloned on the same plasmid. Their attempt to place a promoter for T7 RNA polymerase into a plasmid along with the T7 Gene 1 produced only arrangements in which the promoter directed transcription opposite to the direction needed to transcribe the polymerase gene.
Thus, the art appreciates the potential benefits of placing a foreign gene under the control of a T7 phage promoter. To date such a convenient non-lethal method for providing the T7 RNA polymerase to a system comprising a T7 promoter does not exist. The present invention provides a solution to this problem.